Spinal cord injury (SCI) is accompanied by well-described changes in bladder function. Our previous studies revealed that SCI causes significant disruption to the rodent bladder uro-epithelium (UT) acutely (within hours) and chronically. There is substantive evidence that the urothelium plays a prominent role in the bladder ‘sensory web’, which impacts underlying bladder neural pathways and in turn voiding function. Thus, a better understanding of both early and late phases of SCI-induced urothelial perturbation could prevent the plastic changes in these neural/non-neuronal pathways and, in turn, the development of neurogenic bladder overactivity. Though a prominent consequence of SCI is increased oxidative stress and mitochondrial dysfunction, how these changes affect bladder function following SCI is unknown. Our recent findings show that SCI leads to significant morphological and functional defects in UT mitochondria which can be mitigated by antioxidant treatment. These findings lead us to hypothesize that SCI results in a UT mitochondrial dysregulation that can impair signalling in the UT and bladder wall.  Because a local UT-afferent signalling pathway regulates sensory input to the central nervous system, it is likely that these changes in UT-neural signalling can influence voiding behaviour.  
The aim of this study was to examine the impact of acute versus chronic SCI on urothelial mitochondrial dysregulation/oxidative stress and whether mitochondrial-targeted antioxidant treatment can reverse SCI-induced urothelial defects.

This aim was investigated in female C57 mice (C57Bl/6; 20-25 gm; 4-5 weeks old) with complete spinal cord transection at the T9-T10 vertebrae level. 
• Two time points post SCI were investigated: early (3 days) and late (28 days) 
• Some animals were treated for 3 or 28 days with the mitochondria ROS scavenger MitoTempo (mTEM; 1mg/kg/day delivered via subcutaneous osmotic pump implanted at the time of SCI).
• Bladders were collected from deeply anesthetized mice and utilized for cell culture, electron microscopy and western blot per previously published methods.
• Cultured urothelial cells (UTC) were loaded with various intracellular dyes to examine functional responses. These included: Dihydrorhodamine 123 - DHR123 (fluorescent indicator of reactive oxygen species - ROS) and Tetramethylrhodamine methyl ester - TMRM (fluorescent indicator of mitochondria membrane potential, Ψm; Fig. 2).

We found that SCI alters UT mitochondrial morphology and function. Transmission electron micrographs revealed significant changes in mitochondrial morphology in early SCI (3 days) versus control with electron dense ‘mitobodies’ (dense spots in the mitochondrial matrix indicative of cellular damage) (Fig. 1). This was accompanied by loss of Ψm (~40-50%; decreased staining of TMRM indicating a loss of viability; Fig 2A), as well as increased ROS production (~20%; Fig 2B) and nitrotyrosine levels (marker of DNA oxidative damage).  Elevated ROS levels are likely to play a role in the early structural changes to the mitochondria, as treatment with mTEM led to a striking decrease in mitobodies (Fig. 1), possibly resulting from an upregulation of mitophagy to remove the damaged MITO. While morphological changes in UT mitochondria had partially recovered in late (28 day) SCI, there was still significant functional impairment in UT mitochondria (loss of Ψm) which was partially reversed with the antioxidant mTEM (Fig. 2).

While a number of factors are likely to contribute to impaired urothelial function after SCI, accumulating evidence suggests that altered cellular metabolism (i.e. mitochondrial functions) plays a key role. Mitochondrial damage is often accompanied by increased ROS production with a concomitant decrease in the ability to clear damaged mitochondria by mitophagy, which has been proposed to be central to the impairment of cellular function in various regulatory systems. The mitochondrial membrane potential Ψm is an important parameter to assess the functional state of these organelles and disturbances in Ψm can increase oxidative stress (a hallmark of SCI). ROS scavengers have been shown to protect the spinal cord and improve motor functions in animal models of CNS trauma. These findings suggest that mitochondrial-targeted antioxidant therapy restores mitochondrial balance via ROS scavenging and upregulation of mitophagy to remove damaged mitochondria. This may be a viable treatment for bladder dysfunction after SCI.

SCI impairs UT structure and function which can impact signalling and result in abnormal urodynamic behaviour. Our novel findings provide new exciting insights into how SCI alters fundamental UT cellular signalling processes, resulting specifically in mitochondrial dysfunction and mitophagy. Treatments that affect oxidative stress can stimulate the mitochondria thus restoring ‘normal’ functions. In summary, mitochondria targeted therapies may hold future promise to restore UT structure and signalling after SCI, which may contribute to improvements in bladder function in these patients.